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1.
Int J Mol Sci ; 24(15)2023 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-37569522

RESUMO

We developed and validated a technology platform for designing and testing peptides inhibiting the infectivity of SARS-CoV-2 spike protein-based pseudoviruses. This platform integrates target evaluation, in silico inhibitor design, peptide synthesis, and efficacy screening. We generated a cyclic peptide library derived from the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the angiotensin-converting enzyme 2 (ACE2) receptor. The cell-free validation process by ELISA competition assays and Surface Plasmon Resonance (SPR) studies revealed that the cyclic peptide c9_05, but not its linear form, binds well to ACE2. Moreover, it effectively inhibited the transduction in HEK293, stably expressing the human ACE2 receptor of pseudovirus particles displaying the SARS-CoV-2 spike in the Wuhan or UK variants. However, the inhibitory efficacy of c9_05 was negligible against the Omicron variant, and it failed to impede the entry of pseudoviruses carrying the B.1.351 (South African) spike. These variants contain three or more mutations known to increase affinity to ACE2. This suggests further refinement is needed for potential SARS-CoV-2 inhibition. Our study hints at a promising approach to develop inhibitors targeting viral infectivity receptors, including SARS-CoV-2's. This platform also promises swift identification and evaluation of inhibitors for other emergent viruses.


Assuntos
COVID-19 , Vírus de RNA , Humanos , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2 , Células HEK293 , Peptídeos/farmacologia , Peptídeos Cíclicos , Biblioteca de Peptídeos , Tecnologia , Ligação Proteica
2.
Viruses ; 13(9)2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34578326

RESUMO

The rapid spread of the pandemic caused by the SARS-CoV-2 virus has created an unusual situation, with rapid searches for compounds to interfere with the biological processes exploited by the virus. Doxycycline, with its pleiotropic effects, including anti-viral activity, has been proposed as a therapeutic candidate for COVID-19 and about twenty clinical trials have started since the beginning of the pandemic. To gain information on the activity of doxycycline against SARS-CoV-2 infection and clarify some of the conflicting clinical data published, we designed in vitro binding tests and infection studies with a pseudotyped virus expressing the spike protein, as well as a clinically isolated SARS-CoV-2 strain. Doxycycline inhibited the transduction of the pseudotyped virus in Vero E6 and HEK-293 T cells stably expressing human receptor angiotensin-converting enzyme 2 but did not affect the entry and replication of SARS-CoV-2. Although this conclusion is apparently disappointing, it is paradigmatic of an experimental approach aimed at developing an integrated multidisciplinary platform which can shed light on the mechanisms of action of potential anti-COVID-19 compounds. To avoid wasting precious time and resources, we believe very stringent experimental criteria are needed in the preclinical phase, including infectivity studies with clinically isolated SARS-CoV-2, before moving on to (futile) clinical trials.


Assuntos
COVID-19/virologia , Interações Hospedeiro-Patógeno , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Fenômenos Fisiológicos Virais/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , COVID-19/metabolismo , Ciclo Celular , Chlorocebus aethiops , Doxiciclina/farmacologia , Células HEK293 , Humanos , Ligação Proteica , SARS-CoV-2/ultraestrutura , Glicoproteína da Espícula de Coronavírus , Transdução Genética , Células Vero
3.
Sci Rep ; 8(1): 12252, 2018 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-30115973

RESUMO

Triple-negative breast cancer (TNBC) is a heterogeneous and aggressive neoplasia lacking the expression of hormonal receptors and human epidermal growth factor receptor-2. Accumulating evidence has highlighted the importance of miRNAs dysregulation in the establishment of cancer programs, but the functional role of many miRNAs remains unclear. The description of miRNAs roles might provide novel strategies for treatment. In the present work, an integrated analysis of miRNA transcriptional landscape was performed (N = 132), identifying the significant down-modulation of miR-342-3p in TNBC, probably because of the aberrant activity of estrogen receptor, which serves as a transcription factor of the miRNA, as demonstrated by a siRNA-knockdown approach. The enhanced expression of miR-342-3p significantly decreased cell proliferation, viability and migration rates of diverse TN cells in vitro. Bioinformatic and functional analyses revealed that miR-342-3p directly targets the monocarboxylate transporter 1 (MCT1), which promotes lactate and glucose fluxes alteration, thus disrupting the metabolic homeostasis of tumor cells. Optical metabolic imaging assay defined a higher optical redox ratio in glycolytic cells overexpressing miR-342-3p. Furthermore, we found that hypoxic conditions and glucose starvation attenuate miR-342-3p expression, suggesting a crosstalk program between these metabolic factors. Consistently, miR-342-3p down-modulation is associated with an increased MCT1 expression level and glycolytic score in human triple negative tumors. Overall, we described for the first time the regulatory activity of miR-342-3p on relevant metabolic carcinogenic pathways in TN breast cancers.


Assuntos
Carcinogênese , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/genética , Transportadores de Ácidos Monocarboxílicos/genética , Simportadores/genética , Neoplasias de Mama Triplo Negativas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Glucose/metabolismo , Glicólise , Homeostase/genética , Humanos , Ácido Láctico/metabolismo , Fosforilação Oxidativa , Receptores de Estrogênio/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo
4.
Cancer Res ; 76(18): 5562-72, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27402080

RESUMO

Organization of cancer cells into endothelial-like cell-lined structures to support neovascularization and to fuel solid tumors is a hallmark of progression and poor outcome. In triple-negative breast cancer (TNBC), PDGFRß has been identified as a key player of this process and is considered a promising target for breast cancer therapy. Thus, we aimed at investigating the role of miRNAs as a therapeutic approach to inhibit PDGFRß-mediated vasculogenic properties of TNBC, focusing on miR-9 and miR-200. In MDA-MB-231 and MDA-MB-157 TNBC cell lines, miR-9 and miR-200 promoted and inhibited, respectively, the formation of vascular-like structures in vitro Induction of endogenous miR-9 expression, upon ligand-dependent stimulation of PDGFRß signaling, promoted significant vascular sprouting of TNBC cells, in part, by direct repression of STARD13. Conversely, ectopic expression of miR-200 inhibited this sprouting by indirectly reducing the protein levels of PDGFRß through the direct suppression of ZEB1. Notably, in vivo miR-9 inhibition or miR-200c restoration, through either the generation of MDA-MB-231-stable clones or peritumoral delivery in MDA-MB-231 xenografted mice, strongly decreased the number of vascular lacunae. Finally, IHC and immunofluorescence analyses in TNBC specimens indicated that PDGFRß expression marked tumor cells engaged in vascular lacunae. In conclusion, our results demonstrate that miR-9 and miR-200 play opposite roles in the regulation of the vasculogenic ability of TNBC, acting as facilitator and suppressor of PDGFRß, respectively. Moreover, our data support the possibility to therapeutically exploit miR-9 and miR-200 to inhibit the process of vascular lacunae formation in TNBC. Cancer Res; 76(18); 5562-72. ©2016 AACR.


Assuntos
MicroRNAs/genética , Neovascularização Patológica/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/biossíntese , Neoplasias de Mama Triplo Negativas/patologia , Animais , Western Blotting , Diferenciação Celular , Células Endoteliais/patologia , Feminino , Imunofluorescência , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos SCID , Neovascularização Patológica/patologia , Reação em Cadeia da Polimerase , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Neoplasias de Mama Triplo Negativas/genética
5.
FEBS Open Bio ; 5: 832-43, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26605137

RESUMO

Sulfurtransferases (Strs) and thioredoxins (Trxs) are members of large protein families. Trxs are disulfide reductases and play an important role in redox-related cellular processes. They interact with a broad range of proteins. Strs catalyze the transfer of a sulfur atom from a suitable sulfur donor to nucleophilic sulfur acceptors in vitro, but the physiological roles of these enzymes are not well defined. Several studies in different organisms demonstrate protein-protein interactions of Strs with members of the Trx family. We are interested in investigating the specificity of the interaction between Str and Trx isoforms. In order to use the bimolecular fluorescence complementation (BiFC), several Str and Trx sequences from Arabidopsis thaliana were cloned into the pUC-SPYNE and pUC-SPYCE split-YFP vectors, respectively. Each couple of plasmids containing the sequences for the putative interaction partners were transformed into Arabidopsis protoplasts and screened using a confocal laser scanning microscope. Compartment- and partner-specific interactions could be observed in transformed protoplasts. Replacement of cysteine residues in the redox-active site of Trxs abolished the interaction signal. Therefore, the redox site is not only involved in the redox reaction but also responsible for the interaction with partner proteins. Biochemical assays support a specific interaction among Strs and certain Trxs. Based on the results obtained, the interaction of Strs and Trxs indicates a role of Strs in the maintenance of the cellular redox homeostasis.

6.
PLoS One ; 10(7): e0131519, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26132116

RESUMO

The natural compound zosteric acid, or p-(sulfoxy)cinnamic acid (ZA), is proposed as an alternative biocide-free agent suitable for preventive or integrative anti-biofilm approaches. Despite its potential, the lack of information concerning the structural and molecular mechanism of action involved in its anti-biofilm activity has limited efforts to generate more potent anti-biofilm strategies. In this study a 43-member library of small molecules based on ZA scaffold diversity was designed and screened against Escherichia coli to understand the structural requirements necessary for biofilm inhibition at sub-lethal concentrations. Considerations concerning the relationship between structure and anti-biofilm activity revealed that i) the para-sulfoxy ester group is not needed to exploit the anti-biofilm activity of the molecule, it is the cinnamic acid scaffold that is responsible for anti-biofilm performance; ii) the anti-biofilm activity of ZA derivatives depends on the presence of a carboxylate anion and, consequently, on its hydrogen-donating ability; iii) the conjugated aromatic system is instrumental to the anti-biofilm activities of ZA and its analogues. Using a protein pull-down approach, combined with mass spectrometry, the herein-defined active structure of ZA was matrix-immobilized, and was proved to interact with the E. coli NADH:quinone reductase, WrbA, suggesting a possible role of this protein in the biofilm formation process.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Cinamatos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Escherichia coli/efeitos dos fármacos , Proteínas Repressoras/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Ésteres do Ácido Sulfúrico/farmacologia , Ânions , Antibacterianos/síntese química , Antibacterianos/química , Biofilmes/crescimento & desenvolvimento , Ácidos Carboxílicos/química , Cinamatos/síntese química , Cinamatos/química , Escherichia coli/enzimologia , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/química , Hidrogênio/química , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Ligação Proteica , Proteínas Repressoras/química , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Ésteres do Ácido Sulfúrico/síntese química , Ésteres do Ácido Sulfúrico/química
7.
J Am Soc Nephrol ; 24(3): 445-55, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23393318

RESUMO

Elevated levels of plasma catecholamines accompany ischemic AKI, possibly contributing the inflammatory response. Renalase, an amine oxidase secreted by the proximal tubule, degrades circulating catecholamines and reduces myocardial necrosis, suggesting that it may protect against renal ischemia reperfusion injury. Here, mice subjected to renal ischemia reperfusion injury had significantly lower levels of renalase in the plasma and kidney compared with sham-operated mice. Consistent with this, plasma NE levels increased significantly after renal ischemia reperfusion injury. Furthermore, renal tubular inflammation, necrosis, and apoptosis were more severe and plasma catecholamine levels were higher in renalase-deficient mice subjected to renal ischemia reperfusion compared with wild-type mice. Administration of recombinant human renalase reduced plasma catecholamine levels and ameliorated ischemic AKI in wild-type mice. Taken together, these data suggest that renalase protects against ischemic AKI by reducing renal tubular necrosis, apoptosis, and inflammation, and that plasma renalase might be a biomarker for AKI. Recombinant renalase therapy may have potential for the prevention and treatment of AKI.


Assuntos
Injúria Renal Aguda/prevenção & controle , Isquemia/prevenção & controle , Monoaminoxidase/farmacologia , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Apoptose/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Isquemia/metabolismo , Isquemia/patologia , Necrose Tubular Aguda/metabolismo , Necrose Tubular Aguda/patologia , Necrose Tubular Aguda/prevenção & controle , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monoaminoxidase/deficiência , Monoaminoxidase/genética , Monoaminoxidase/metabolismo , Infiltração de Neutrófilos/efeitos dos fármacos , Norepinefrina/sangue , Fentolamina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/farmacologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/prevenção & controle
8.
Curr Pharm Des ; 19(14): 2540-51, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23116393

RESUMO

Renalase is a flavoprotein recently discovered in humans, preferentially expressed in the proximal tubules of the kidney and secreted in blood and urine. It is highly conserved in vertebrates, with homologs identified in eukaryotic and prokaryotic organisms. Several genetic, epidemiological, clinical and experimental studies show that renalase plays a role in the modulation of the functions of the cardiovascular system, being particularly active in decreasing the catecholaminergic tone, in lowering blood pressure and in exerting a protective action against myocardial ischemic damage. Deficient renalase synthesis might be the cause of the high occurrence of hypertension and adverse cardiac events in kidney disease patients. Very recently, recombinant human renalase has been structurally and functionally characterized in vitro. Results show that it belongs to the p-hydroxybenzoate hydroxylase structural family of flavoenzymes, contains non-covalently bound FAD with redox features suggestive of a dehydrogenase activity, and is not a catecholamine-degrading enzyme,either through oxidase or NAD(P)H-dependent monooxygenase reactions. The biochemical data now available will hopefully provide the basis for a systematic and rational quest toward the identification of the reaction catalyzed by renalase and of the molecular mechanism of its physiological action, which in turn are expected to favor the development of novel therapeutic tools for the treatment of kidney and cardiovascular diseases.


Assuntos
Catecolaminas/metabolismo , Flavoproteínas/fisiologia , Monoaminoxidase/fisiologia , Transdução de Sinais/fisiologia , Animais , Sítios de Ligação , Doenças Cardiovasculares/enzimologia , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/genética , Descoberta de Drogas , Flavoproteínas/química , Flavoproteínas/genética , Humanos , Nefropatias/enzimologia , Nefropatias/epidemiologia , Nefropatias/genética , Monoaminoxidase/química , Monoaminoxidase/genética , Polimorfismo de Nucleotídeo Único , Conformação Proteica , Fatores de Risco
9.
Biochemistry ; 51(18): 3819-26, 2012 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-22519987

RESUMO

Plasmodium falciparum ferredoxin-NADP(+) reductase (FNR) is a FAD-containing enzyme that, in addition to be a promising target of novel antimalarial drugs, represents an excellent model of plant-type FNRs. The cofactor specificity of FNRs depends on differences in both k(cat) and K(m) values for NADPH and NADH. Here, we report that deletion of the hydroxyl group of the conserved Y258 of P. falciparum FNR, which interacts with the 2'-phosphate group of NADPH, selectively decreased the k(cat) of the NADPH-dependent reaction by a factor of 2 to match that of the NADH-dependent one. Rapid-reaction kinetics, active-site titrations with NADP(+), and anaerobic photoreduction experiments indicated that this effect may be the consequence of destabilization of the catalytically competent conformation of bound NADPH. Moreover, because the Y258F replacement increased the K(m) for NADPH 4-fold and decreased that for NADH 3-fold, it led to a drop in the ability of the enzyme to discriminate between the coenzymes from 70- to just 1.5-fold. The impact of the Y258F change was not affected by the presence of the H286Q mutation, which is known to enhance the catalytic activity of the enzyme. Our data highlight the major role played by the Y258 hydroxyl group in determining the coenzyme specificity of P. falciparum FNR. From the general standpoint of engineering the kinetic properties of plant-type FNRs, although P. falciparum FNR is less strictly NADPH-dependent than its homologues, the almost complete abolishment of coenzyme selectivity reported here has never been accomplished before through a single mutation.


Assuntos
Ferredoxina-NADP Redutase/química , NADP/metabolismo , Tirosina/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Coenzimas/metabolismo , Ferredoxina-NADP Redutase/genética , Ferredoxina-NADP Redutase/metabolismo , Cinética , NAD/metabolismo , Plasmodium falciparum/enzimologia , Especificidade por Substrato
10.
J Mol Biol ; 411(2): 463-73, 2011 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-21699903

RESUMO

Renalase is a recently discovered flavoprotein that regulates blood pressure, regulates sodium and phosphate excretion, and displays cardioprotectant action through a mechanism that is barely understood to date. It has been proposed to act as a catecholamine-degrading enzyme, via either O(2)-dependent or NADH-dependent mechanisms. Here we report the renalase crystal structure at 2.5 Å resolution together with new data on its interaction with nicotinamide dinucleotides. Renalase adopts the p-hydroxybenzoate hydroxylase fold topology, comprising a Rossmann-fold-based flavin adenine dinucleotide (FAD)-binding domain and a putative substrate-binding domain, the latter of which contains a five-stranded anti-parallel ß-sheet. A large cavity (228 Å(3)), facing the flavin ring, presumably represents the active site. Compared to monoamine oxidase or polyamine oxidase, the renalase active site is fully solvent exposed and lacks an 'aromatic cage' for binding the substrate amino group. Renalase has an extremely low diaphorase activity, displaying lower k(cat) but higher k(cat)/K(m) for NADH compared to NADPH. Moreover, its FAD prosthetic group becomes slowly reduced when it is incubated with NADPH under anaerobiosis, and binds NAD(+) or NADP(+) with K(d) values of ca 2 mM. The absence of a recognizable NADP-binding site in the protein structure and its poor affinity for, and poor reactivity towards, NADH and NADPH suggest that these are not physiological ligands of renalase. Although our study does not answer the question on the catalytic activity of renalase, it provides a firm framework for testing hypotheses on the molecular mechanism of its action.


Assuntos
Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Monoaminoxidase/química , Monoaminoxidase/metabolismo , NADP/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , NAD/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
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